The objective of the proposed research is to elucidate the mechanism of antidiuretic hormone (ADH) action, through mediation of cyclic AMP (cAMP) on kidney in health and disease. Major focus will be aimed to investigate how cAMP regulates water permeability of collecting tubules of mammalian nephron. Specific objectives are to investigate: a) Components of cAMP-mediated cellular ADH action in isolated microdissected collecting tubules and in the ascending limbs of Henle's loops: adenylate cyclase, cAMP phosphodiesterase, components of cAMP-dependent protein phosphorylations, microtubules, microfilaments (and other contractile and cytoskeletal elements) and luminal plasma membrane with associated structures. b) To develop methods for isolation and characterization of luminal plasma membrane of mammalian collecting tubules; to evaluate and determine properties of luminal plasma membrane of collecting tubules in relation to cAMP action. c) To determine intracellular localization in tubules of cAMP, protein kinase, protein phosphatase, microtubules, microfilaments and related components of ADH action namely in cells of collecting tubules and ascending limbs of Henle's loop. d) To investigate cellular mechanism of ADH action in animal models of renal hyporesponsiveness (nephrogenic diabetes insipidus) and hyperresponsiveness to ADH, spontaneous or induced by drugs. These studies will be conducted on microdissected segments of nephron and will also include intracellular localization studies. Studies will be conducted on individual microdissected tubule segments, namely collecting tubules and ascending limb of Henle's loop or cell populations derived from these segments in order to determine basic components of cellular ADH action, their contribution to the renal ADH-dependent concentrating mechanism and loci of impairment in the abnormal response to kidney to ADH.